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Pull down assay kit

Manufactured by Cytoskeleton
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The Pull-down assay kit is a laboratory tool used to study protein-protein interactions. It allows the identification and isolation of proteins that bind to a specific target protein of interest. The kit includes all the necessary components and reagents to perform the pull-down assay procedure.

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Lab products found in correlation

Antibody for β actin, by Merck Group (2 mentions) Mem per plus membrane extraction kit, by Thermo Fisher Scientific (2 mentions) P rex1 antibody, by R&D Systems (2 mentions) Rat high range insulin elisa, by ALPCO (2 mentions) P rex1sirna smartpool, by Horizon Discovery (2 mentions) Rac1 antibody, by Merck Group (2 mentions) Dharmafect 1, by Horizon Discovery (2 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) E cadherin, by Cell Signaling Technology (2 mentions) G lisa, by Cytoskeleton (1 mentions)

5 protocols using pull down assay kit

1

Quantifying RAC1 Activation in Retinal Microvessels

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RAC1 activation was determined by the pull-down assay kit (Cytoskeleton, Denver, CO, USA) and the relative amount of activated RAC1 (RAC1-GTP) was quantified by western blotting and densitometry [29 (link), 30 (link)]. RAC1 activation in human retinal microvessels was quantified using a G-LISA (Cytoskeleton).
Kowluru R.A., Kowluru A., Veluthakal R., Mohammad G., Syed I., Santos J.M, & Mishra M. (2014). TIAM1–RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy. Diabetologia, 57(5), 1047-1056.
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2

Rac1 Activity Assay in Cadm1 MEFs

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The level of active Rac1 was assayed using a pull-down assay kit (cytoskeleton, Denver, CO, USA) following the manufacturer's instructions. Briefly, vGPCR expressing Cadm1+/+ and Cadm1−/− MEFs were harvested and lysed in lysis buffer. Equal amounts of lysates were incubated with PAK-PBD beads at 4°C for 2 h. PAK-PBD beads were pelleted by centrifugation and washed with wash buffer. Active Rac1 was detected by western blotting.
Hunte R., Alonso P., Thomas R., Bazile C.A., Ramos J.C., van der Weyden L., Dominguez-Bendala J., Khan W.N, & Shembade N. (2018). CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation. PLoS Pathogens, 14(4), e1006968.
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3

Investigating P-Rex1 and RhoG Signaling

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P-Rex1 antibody was from R&D Systems (Minneapolis, MN, USA). Antisera directed against RhoG was from Santa Cruz Biotechnology (CA, USA). E-Cadherin, GAPDH and HRP-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA). Rac1 antibody was from EMD Millipore (Burlington, MA, USA). Rat high range insulin ELISA was from ALPCO (Salem, NH, USA). Target sequence for the ON-TARGETplus Non-targeting siRNA#1 (Catalog Item- D-001810–01-20) is: UGGUUUACAUGUCGACUAA. The target sequences for the ON-TARGETplus Rat Prex1(311647) siRNA-SMARTpool (Catalog Item L-093719–02-0010 that has 4 sequences that it targets) are: GCAUGGAGCGCGACGCAUA, CAACAACAACGGCGAGUAU, UCCUGAAAGUCAACGGCAA and CCAUCAACGCCCUGGACGA. The above on-target P-Rex1siRNA SMARTpool and non-targeting control siRNA (Con-si), as well as DharmaFect1, were from Dharmacon (Lafayette, CO, USA). Antibody for β-actin and all other reagents used in the current studies were from Sigma Aldrich (St. Louis, MO, USA). Mem-PER Plus Membrane Extraction Kit was from Thermo Fisher Scientific (Waltham, MA, USA). The pull down assay kit used for the Rac1 activation was from Cytoskeleton (Denver, CO, USA).
Thamilselvan V., Gamage S., Harajli A., Chundru S.A, & Kowluru A. (2020). P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(6), 1218-1230.
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4

Detecting Activated RhoA by Western Blotting

Cited 1 time
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Western blotting was performed as previously described30 (link), 34 (link). A list of primary and secondary antibodies, including manufacturer number and working concentration, may be found in Supplementary Table 1. Immunoprecipitation to detect activated RhoA was performed using a pulldown assay kit (Cytoskeleton, Inc.) according to manufacturer instructions.
Burke R.M., Lighthouse J.K., Mickelsen D.M, & Small E.M. (2019). Sacubitril/valsartan decreases cardiac fibrosis in left ventricle pressure overload by restoring PKG signaling in cardiac fibroblasts. Circulation. Heart failure, 12(4), e005565.
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5

Investigating P-Rex1 and RhoG Signaling

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P-Rex1 antibody was from R&D Systems (Minneapolis, MN, USA). Antisera directed against RhoG was from Santa Cruz Biotechnology (CA, USA). E-Cadherin, GAPDH and HRP-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA). Rac1 antibody was from EMD Millipore (Burlington, MA, USA). Rat high range insulin ELISA was from ALPCO (Salem, NH, USA). Target sequence for the ON-TARGETplus Non-targeting siRNA#1 (Catalog Item- D-001810–01-20) is: UGGUUUACAUGUCGACUAA. The target sequences for the ON-TARGETplus Rat Prex1(311647) siRNA-SMARTpool (Catalog Item L-093719–02-0010 that has 4 sequences that it targets) are: GCAUGGAGCGCGACGCAUA, CAACAACAACGGCGAGUAU, UCCUGAAAGUCAACGGCAA and CCAUCAACGCCCUGGACGA. The above on-target P-Rex1siRNA SMARTpool and non-targeting control siRNA (Con-si), as well as DharmaFect1, were from Dharmacon (Lafayette, CO, USA). Antibody for β-actin and all other reagents used in the current studies were from Sigma Aldrich (St. Louis, MO, USA). Mem-PER Plus Membrane Extraction Kit was from Thermo Fisher Scientific (Waltham, MA, USA). The pull down assay kit used for the Rac1 activation was from Cytoskeleton (Denver, CO, USA).
Thamilselvan V., Gamage S., Harajli A., Chundru S.A, & Kowluru A. (2020). P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 54(6), 1218-1230.
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